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Zika Virus in Gabon (Central Africa) – 2007: A New Threatfrom Aedes albopictus?Gilda Grard1*, Melanie Caron1,2, Illich Manfred Mombo1,2, Dieudonne Nkoghe1,3, Statiana Mboui Ondo1,
Davy Jiolle2,4, Didier Fontenille2, Christophe Paupy2,4, Eric Maurice Leroy1,2
1 UMVE, Centre International de Recherches Medicales de Franceville, Franceville, Gabon, 2 MIVEGEC, Institut de Recherche pour le Developpement (IRD-224, CNRS-5290,
Universites de Montpellier 1 & 2), Montpellier, France, 3 Ministere de la Sante Publique, Libreville, Gabon, 4 URES, CIRMF, Franceville, Gabon
Abstract
Background: Chikungunya and dengue viruses emerged in Gabon in 2007, with large outbreaks primarily affecting thecapital Libreville and several northern towns. Both viruses subsequently spread to the south-east of the country, with newoutbreaks occurring in 2010. The mosquito species Aedes albopictus, that was known as a secondary vector for both viruses,recently invaded the country and was the primary vector involved in the Gabonese outbreaks. We conducted aretrospective study of human sera and mosquitoes collected in Gabon from 2007 to 2010, in order to identify othercirculating arboviruses.
Methodology/Principal Findings: Sample collections, including 4312 sera from patients presenting with painful febriledisease, and 4665 mosquitoes belonging to 9 species, split into 247 pools (including 137 pools of Aedes albopictus), werescreened with molecular biology methods. Five human sera and two Aedes albopictus pools, all sampled in an urban settingduring the 2007 outbreak, were positive for the flavivirus Zika (ZIKV). The ratio of Aedes albopictus pools positive for ZIKVwas similar to that positive for dengue virus during the concomitant dengue outbreak suggesting similar mosquitoinfection rates and, presumably, underlying a human ZIKV outbreak. ZIKV sequences from the envelope and NS3 geneswere amplified from a human serum sample. Phylogenetic analysis placed the Gabonese ZIKV at a basal position in theAfrican lineage, pointing to ancestral genetic diversification and spread.
Conclusions/Significance: We provide the first direct evidence of human ZIKV infections in Gabon, and its first occurrencein the Asian tiger mosquito, Aedes albopictus. These data reveal an unusual natural life cycle for this virus, occurring in anurban environment, and potentially representing a new emerging threat due to this novel association with a highly invasivevector whose geographic range is still expanding across the globe.
Citation: Grard G, Caron M, Mombo IM, Nkoghe D, Ondo SM, et al. (2014) Zika Virus in Gabon (Central Africa) – 2007: A New Threat from Aedes albopictus? PLoSNegl Trop Dis 8(2): e2681. doi:10.1371/journal.pntd.0002681
Editor: Remi Charrel, Aix Marseille University, France
Received June 27, 2013; Accepted December 19, 2013; Published February 6, 2014
Copyright: � 2014 Grard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by CIRMF, which is funded by the Gabonese Government, Total Gabon and the French Foreign Ministry. It was also partiallyfinanced by grants from Fondation Christophe et Rodolphe Merieux, Institut de France, Institut de recherches pour le developpement (IRD),and Metabiota. Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gildagrard@gmail.com
Introduction
Zika virus (ZIKV) is a mosquito-borne flavivirus phylogeneti-
cally related to dengue viruses. Following its first isolation in 1947
from a sentinel monkey placed in the Zika forest in Uganda [1],
serological surveys and viral isolations (reviewed in [2]) suggested
that ZIKV (i) ranged widely throughout Africa and Asia, and (ii)
circulated according to a zoonotic cycle involving non-human
primates and a broad spectrum of potential mosquito vector
species.
In Africa, ZIKV has been isolated from humans in western and
central countries such as Senegal, Nigeria, Central African
Republic and Uganda [3–7]. Serological surveys (reviewed in
[2]) suggested that its geographic range might extend not only to
other West and Central African countries (Sierra Leone,
Cameroon, Gabon), but also to eastern (Ethiopia, Kenya,
Tanzania and Somalia) and northern Africa (Egypt). ZIKV has
also been isolated from mosquitoes collected in Senegal, Ivory
Coast, Burkina Faso, Central African Republic and Uganda
[1,6,8,9]. These mosquitoes mainly belonged to sylvan or rural
species of the genus Aedes, and more precisely to the Aedimorphus,
Diceromyia and Stegomyia subgenera. The virus has also been isolated
in West Africa (Burkina Faso, Senegal and Ivory Coast) [6,9] and
Asia [10] from Aedes aegypti, a species being considered the main
ZIKV epidemic vector outside Africa [11]. Moreover, Ae. aegypti
was shown experimentally to be an efficient ZIKV vector [12–14].
Despite its apparent broad geographic distribution in Africa and
Asia, only sporadic cases of human ZIKV infection have been
reported. This virus received little attention until its sudden
emergence in Yap Island (Micronesia) in 2007, which involved
about 5000 persons [15,16], revealing its epidemic capacity.
Patients develop a mild dengue-like syndrome, including fever,
headache, rash, arthralgia and conjunctivitis. This clinical similarity
with other, more commonly diagnosed arboviral infections such as
chikungunya (CHIKV) and dengue (DENV), might delay the
diagnosis and/or lead to underestimation of ZIKV infections.
PLOS Neglected Tropical Diseases | www.plosntds.org 1 February 2014 | Volume 8 | Issue 2 | e2681
Here, we report the first direct evidence of ZIKV epidemic
activity in Central Africa, and its occurrence in an urban
environment during concomitant CHIKV/DENV outbreaks in
Libreville, the capital of Gabon, in 2007. We also report the first
detection of ZIKV in the Asian tiger mosquito, Ae. albopictus. These
findings, together with the global geographic expansion of this
invasive species and its increasing importance as epidemic vector
of arboviruses as exemplified by CHIKV adaptation, suggest that
the prerequisites for the emergence and global spread of Zika virus
may soon be satisfied.
Materials and Methods
StudyIn 2007 and 2010, Gabon recorded simultaneous outbreaks of
CHIKV (genus Alphavirus) and DENV (genus Flavivirus) infections.
The 2007 outbreaks primarily affected Libreville, the capital of
Gabon, and subsequently extended northwards to several other
towns [17], while the 2010 outbreaks occurred in the south-eastern
provinces [18]. To detect other circulating arboviruses, we
conducted a retrospective study based on molecular screening of
4312 sera from symptomatic patients presenting to healthcare
centers; 24.7% of the samples were obtained during the 2007
outbreaks, 9.7% during the inter-epidemic period, and 65.5%
during the 2010 outbreaks (data not shown). We also analyzed a
collection of 4665 mosquitoes captured during the same period
and split into 247 pools according to the species, date and
sampling site (Table 1, see [18] and [19] for the details of the
methodology used for mosquito trapping).
Ethics statementThe Centre International de Recherches Medicales de France-
ville (CIRMF) and the Gabonese Ministry of Health cooperated in
the 2007 and 2010 outbreak response and management, that
included blood sampling for laboratory diagnostic and epidemi-
ological survey. The study was approved by our Institutional
review board (Conseil scientifique du CIRMF).
Symptomatic patients presented to health care centers for
medical examination. All patients were informed that blood
sampling was required for laboratory diagnosis of suspected acute
infections, such as malaria, dengue or chikungunya fever. During
the two outbreaks, given the urgency of diagnosis, only oral
consent was obtained for blood sampling and was approved by the
institutional review board. However during the active surveillance
study that was performed between the two outbreaks (described in
reference [18]), written consent could be obtained.
Virus identification and characterizationPrimary molecular screening was based on hemi-nested reverse-
transcription PCR (hnRT-PCR) with the generic primers PF1S/
PF2Rbis/PF3S targeting highly conserved motifs in the flavivirus
polymerase (NS5) gene (280-bp) [20]. Yellow fever virus RNA
(vaccinal strain 17D) was used as a positive control. A second
screening was performed with a ZIKV-specific real-time PCR
method using the primers-probe system ZIKV-1086/ZIKV-
1162c/ZIKV-1107-FAM [16], also targeting a short sequence
(160 bp) of the NS5 gene.
Virus isolation was attempted on the Vero and C6/36 cell lines
but was unsuccessful, presumably because of low viral titers
(despite two patients presenting only 1 and 4 days after symptom
onset), and unsuitable initial storage conditions. To further
characterize the Gabonese ZIKV strains, partial envelope (E)
(841 bp) and NS3 (772 bp) gene sequences were amplified by
conventional nested RT-PCR with specific primers derived from
published ZIKV sequences. The primer pairs targeting the E gene
were ZIK-ES1 (TGGGGAAAYGGDTGTGGACTYTTTGG)/
ZIK-ER1 (CCYCCRACTGATCCRAARTCCCA) and ZIK-
ES2 (GGGAGYYTGGTGACATGYGCYAAGTT)/ZIK-ER2
(CCRATGGTGCTRCCACTCCTRTGCCA). The primer pairs
for NS3 amplification were ZIK-NS3FS (GGRGTCTTCCACA-
CYATGTGGCACGTYACA)/ZIK-NS3FR (TTCCTGCCTAT-
RCGYCCYCTCCTCTGRGCAGC) and ZIK-X1 (AGAGTGA-
TAGGACTCTATGG)/ZIK-X2 (GTTGGCRCCCATCTCT-
GARATGTCAGT).
Phylogenetic analysisThe E and NS3 sequences obtained from one Gabonese patient
were concatenated and analyzed using a set of previously
published ZIKV sequences. Phylogenetic relationships were
reconstructed with the maximum likelihood algorithm implement-
ed in PhyML [21] (available at http://www.atgc-montpellier.fr/
phyml/) with best of NNI (Nearest Neighbor Interchange) and
SPR (Subtree Pruning and Regrafting) criteria for tree topology
searching, and the GTR model of nucleotide substitutions. The
Gamma distribution of rate heterogeneity was set to 4 categories,
with a proportion of invariable sites and an alpha parameter
estimated from the dataset. Branch support was assessed from 100
bootstrap replicates. Tree reconstructions were also performed by
Bayesian inference with MrBayes v3.2 [22] under the GTR+I+G
model of nucleotide substitutions, and with the distance neighbor-
joining method [23] implemented in MEGA5 [24] with
confidence levels estimated for 1000 replicates. To test for
phylogenetic discrepancies, tree reconstructions were also per-
formed independently from the envelope dataset and the NS3
dataset with PhyML according to the parameters described above.
The resulting trees were visualized with the FigTree software
(Available at: http://tree.bio.ed.ac.uk/software/figtree/), and
rooted on midpoint for clarity. The Genbank accession numbers
for the Gabonese ZIKV strain are KF270886 (envelope) and
KF270887 (NS3).
Author Summary
Not previously considered an important human arboviralpathogen, the epidemic capacity of Zika virus (ZIKV, adengue-related flavivirus) was revealed by the Micronesiaoutbreak in 2007, which affected about 5000 persons.Widely distributed throughout tropical areas of Asia andAfrica, ZIKV is transmitted by a broad range of mosquitospecies, most of which are sylvatic or rural, Aedes aegypti,an anthropophilic and urban species, being considered themain ZIKV epidemic vector. In a context of emergingarbovirus infections (chikungunya (CHIKV) and dengue(DENV)) in Gabon since 2007, we conducted a retrospec-tive study to detect other, related viruses. In samplescollected during the concurrent CHIKV/DENV outbreaksthat occurred in the capital city in 2007, we detected ZIKVin both humans and mosquitoes, and notably the Asianmosquito Aedes albopictus that recently invaded thecountry and was the main vector responsible for theseoutbreaks. We found that the Gabonese ZIKV strainbelonged to the African lineage, and phylogenetic analysissuggested ancestral diversification and spread rather thanrecent introduction. These findings, showing for the firsttime epidemic ZIKV activity in an urban environment inCentral Africa and the presence of ZIKV in the invasivemosquito Aedes albopictus, raise the possibility of a newemerging threat to human health.
Aedes albopictus Transmission of Zika Virus, Gabon
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Results
Molecular screeningThe NS5 PCR products were sequenced, resulting in the first
ZIKV RNA detection in a human sample (Cocobeach) and in two
Ae. albopictus pools (Libreville) collected during the 2007 outbreaks.
Real-time PCR was then performed, leading to the detection of
four additional positive human samples, collected in 2007 in four
suburbs of Libreville (Diba-Diba, Nzeng-Ayong, PK8, PK9)
(Figure 1). No ZIKV was detected during the inter-epidemic
period or during the 2010 outbreaks.
Clinical descriptionClinical information was available for only one ZIKV-positive
patient, who had mild arthralgia, subjective fever, headache, rash,
mild asthenia, myalgia, diarrhea and vomiting. No information
was available on this patient’s outcome. Cycle threshold values for
human blood samples were high (.37 cycles), suggesting low viral
loads (data not shown).
Vector involvementAedes albopictus was the predominant species collected, account-
ing for 55.4% of the mosquito pools, while Aedes aegypti accounted
for 18.2% (Table 1). The other mosquito species consisted of
members of the Aedes simpsoni complex, Anopheles gambiae, Mansonia
africana, Mansonia uniformis, Culex quinquefasciatus, Eretmapodites
quinquevittatus and unidentified Culex species. Positive mosquito
pools were captured from two suburbs (Nzeng-Ayong and
Alenkiri) where Aedes albopictus was the predominant species
(Figure 1, Table 1).
Sequences analysisAs isolation on the Vero and C6/36 cell lines failed, the
Gabonese ZIKV strain was further characterized by partial
sequencing of the E and NS3 genes. Phylogenetic analysis was
performed on concatenated E and NS3 sequences from one
Cocobeach serum sample. The resulting tree topology (Figure 2)
was similar to that previously obtained from the complete coding
sequences, corroborating Asian and African distinct lineages [2].
The African lineage was further split into two groups, one
containing the genetic variants of the MR766 strain (Uganda,
1947) and the second including West African strains (Nigeria,
1968; Senegal, 1984) and the new ZIKV sequence from Gabon, at
a basal position. Phylogenetic trees derived from the E and NS3
partial sequences resulted in a similar topology, apart from the
weakly supported branching pattern for the MR766 variant
DQ859059, oscillating between the two African sister groups
(Supporting Figure S1). The deletions in potential glycosylation
sites previously reported for the Nigerian ZIKV strain and two
variants of the Ugandan strain MR766 (sequences AY632535 and
DQ859059) [2] were absent from the Gabonese ZIKV sequence.
Discussion
Evidence of human ZIKV infections in Central Africa is limited to
one isolate from RCA in 1991 [6] and two serological surveys
Table 1. Mosquito collections screened for Zika virus.
Libreville 2007Franceville2010 Total
Species Pools Mos. Id. (No.) ZIKV CHIKV DENVLibrevillesuburb Pools Mos. Pools (%) Mos.
Aedes albopictus 91 2130 T64 (21) + + 2 Nzeng-Ayong 46 571 137 (55.4) 2701
T713 (25) + + 2 Alenkiri
T707 (25) 2 2 + Alenkiri
T717 (25) 2 2 + Alenkiri
T723 (25) 2 2 + Alenkiri
T724 (6) 2 + 2 Alenkiri
T21 (25) 2 + 2 Avorembam
T22 (25) 2 + 2 Avorembam
T280 (1) 2 + 2 Bel-Air
Aedes aegypti* 40 853 5 28 45 (18.2) 881
Aedes simpsonicomplex
10 52 5 36 15 (6.1) 88
Anopheles gambiae* 8 72 8 (3.2) 72
Mansonia africana 6 86 6 (2.4) 86
Mansonia uniformis* 4 99 4 (1.6) 99
Culex quinquefasciatus 29 690 29 (11.7) 690
Culex spp. 1 22 1 (0.4) 22
Eretmapoditesquinquevittatus*
2 26 2 (0.8) 26
Total 189 4004 58 661 247 4665
*Species in which Zika virus has previously been detected.(%) The percentage of each mosquito species in the collection is indicated in brackets. Mos.: Number of mosquitoes included in a pool. Id. (No.): Mosquito pool positivefor ZIKV, CHIKV or DENV, followed by the total number of included mosquitoes in the pool indicated in brackets.doi:10.1371/journal.pntd.0002681.t001
Aedes albopictus Transmission of Zika Virus, Gabon
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performed 50 years ago in Gabon [25,26]. No report of human
ZIKV infections was made in other countries of the Congo basin
forest block, despite probable circulation through a sylvan natural
cycle. We provide here the first direct evidence of human ZIKV
infections in Gabon, as well as its occurrence in an urban transmission
cycle, and the probable role of Ae. albopictus as an epidemic vector.
Figure 1. Geographic distribution of Zika and chikungunya and/or dengue viruses infections in Gabon in 2007. The left-hand panelindicates Gabonese CHIKV and/or DENV cases in green circles and ZIKV cases in purple circles. The right-hand panel shows the location of Librevillesuburbs where ZIKV-positive human sera (H) and mosquito pools (M) were detected.doi:10.1371/journal.pntd.0002681.g001
Figure 2. Phylogenetic relationships between concatenated sequences of the Zika virus envelope and NS3 genes. The tree wasconstructed with the maximum likelihood algorithm implemented in PhyML and rooted on midpoint. Bootstrap values are shown at the respectivenodes, followed by bootstrap values resulting from NJ analysis and, finally, the posterior probability resulting from Bayesian analysis. The scale barindicates the number of substitutions per site. The GenBank accession numbers for the 2007 Gabonese ZIKV isolate are KF270886 (envelope) andKF270887 (NS3).doi:10.1371/journal.pntd.0002681.g002
Aedes albopictus Transmission of Zika Virus, Gabon
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Our phylogenetic results are in agreement with the tree topology
previously obtained with complete coding sequences of ZIKV strains,
showing an African lineage and an Asian lineage [2]. The branching
pattern obtained here suggests that ZIKV emergence in Gabon did
not result from strain importation but rather from the diversification
and spread of an ancestral strain belonging to the African lineage.
The identification of ZIKV in two different localities of Gabon
(Cocobeach and Libreville) suggests that the virus was widespread
rather than restricted to a single epidemic focus. The simultaneous
occurrence of human and mosquito infections in Libreville also
suggests that the virus circulated in 2007 in an epidemic cycle rather
than as isolated cases introduced from sylvan cycles.
Of note, ZIKV transmission occurred here in a previously
undocumented urban cycle, supporting the potential for urbani-
zation suggested in 2010 by Weaver and Reisen [27]. While some
mosquito species (including Ae. aegypti) previously found to be
associated with ZIKV, were captured and tested here, only Ae.
albopictus pools were positive for this virus. Moreover, this species
largely outnumbered Ae. aegypti in the suburbs of Libreville where
human cases were detected, suggesting that Ae. albopictus played a
major role in ZIKV transmission in Libreville.
The ratio of ZIKV-positive Ae. albopictus pools is similar to that
reported for DENV-positive pools, suggesting that these two
viruses infect similar proportions of mosquitoes. The small number
of recorded human ZIKV cases, by comparison with DENV cases,
may be due to the occurrence of subclinical forms of ZIKV
infections that did not required medical attention. Thus, an
underlying ZIKV epidemic transmission might have been masked
by concomitant CHIKV/DENV outbreaks.
The natural histories of CHIKV and ZIKV display several
similarities. Before the large Indian Ocean outbreaks in 2005–2007,
chikungunya fever was a neglected arboviral disease. Both viruses
are phylogenetically closely related to African viruses [28–30]
suggesting they probably originated in Africa, where they circulated
in an enzootic sylvan cycle involving non-human primates and a
wide variety of mosquito species, human outbreaks presumably
being mediated by Ae. aegypti [5,31]. In Asia, both viruses are
thought to circulate mainly in a human-mosquito cycle involving Ae.
aegypti [11,14,31]. Together with the recent Yap Island outbreak,
this prompted some researchers to re-examine the susceptibility of
Ae. aegypti to ZIKV infection [14]. However, it must be noted that
the vector of the Yap Island outbreak was not definitely identified
since the predominant potential vector species Aedes hensilli remained
negative [15], and that ZIKV has been isolated only once from Ae.
aegypti in Asia [10], so that its vector status in natura is not confirmed.
Additionally, a ZIKV enzootic transmission cycle involving non-
human primates in Asia and sylvatic vectors cannot be ruled out as
suggested by serologic studies carried on orangutang [32,33].
Finally both CHIKV and ZIKV have shown their ability to adapt to
a new vector, Ae. albopictus, upon introduction in an environment
where their primary vector was outnumbered. This mosquito
species being native to South-East Asia, our findings may help to
explain human ZIKV transmission in Asia.
Aedes albopictus was first introduced in Africa in 1991 [34] and
detected in Gabon in 2007, where its invasion likely contributed to
the emergence of CHIKV and DENV in this country [17–19,35].
Multiple lines of evidence supporting its increasing impact as an
arboviral vector have also been obtained during CHIKV outbreaks
in the Indian Ocean region (2005–2007) and in Italy (2007) [36,37]
through viral evolutionary convergence of Ae. albopictus adaptive
mutations [38–41]. Whether or not the transmission of ZIKV in
Central Africa was also link to such an adaptative mutation of ZIKV
to Ae. albopictus cannot be answered at this stage. Wong and
colleagues [42] have just demonstrated experimentally that Ae.
albopictus strains from Singapore were orally receptive to the
Ugandan strain of ZIKV sampled in 1947, suggesting that this
virus-vector association in Africa may have been previously
prevented because the required ecological conditions did not yet
exist. However, given the relatively low ZIKV viral loads previously
reported in patients - with an order of magnitude of 105 copies/ml
compared to 107 to 109 copies/ml for CHIKV [16,18,40] - the oral
infectivity for Ae. albopictus may seem at least as critical as it was for
CHIKV in establishing this new human-mosquito cycle.
Why ZIKV has not yet been detected in the areas where DENV
and CHIKV have already spread via Ae. albopictus is unclear, but it
may be an ongoing process which we are just starting to detect.
The spread of CHIKV reflects the ability of arboviruses to adapt
to alternative hosts, and the resulting public health concerns in
both developed and developing countries. Is ZIKV the next virus
to succeed CHIKV as an emerging global threat? The increasing
geographic range of Ae. albopictus in Africa, Europe, and the
Americas [34,36,43,44], together with the ongoing ZIKV
outbreak in French Polynesia at the time of writing [45] suggest
this possibility should be seriously considered. Analysis of sylvan
and urban transmission cycles, together with viral genetics and
vector competence studies, are now required to assess (i) how
ZIKV is able to establish a sustainable transmission cycle involving
this new vector in Central Africa, (ii) vector(s)-virus relationships in
Asia, and (iii) the risk of importation and spread to new areas
where Ae. albopictus occurs as well.
Supporting Information
Figure S1 Phylogenetic trees reconstructed from the E and NS3
datasets. Analyses were performed with the maximum likelihood
algorythm implemented in PhyML and parameters set as
described in the Methods section. Trees are rooted on midpoint.
(TIF)
Acknowledgments
We thank Phillippe Yaba, Andre Delicat, Philippe Engandja, Boris
Makanga and Judicael Obame Nkoghe from CIRMF for their technical
assistance.
Author Contributions
Conceived and designed the experiments: GG MC DF CP EML.
Performed the experiments: GG MC IMM DJ SMO. Analyzed the data:
GG MC DF CP EML. Contributed reagents/materials/analysis tools: GG
MC IMM SMO DJ DN. Wrote the paper: GG MC CP EML.
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PLOS Neglected Tropical Diseases | www.plosntds.org 6 February 2014 | Volume 8 | Issue 2 | e2681